The goal of this proposal is to determine how the Notch signaling pathway regulates the formation and growth of brain tumors, and to develop new brain tumor therapies based on Notch pathway inhibition. Notch receptors control the specification, proliferation and survival of stem and progenitor cells in the brain. Notch signaling is also implicated in tumorigenesis, including the formation and growth of brain tumors such as medulloblastoma and glioma. We have recently shown that the Notch2 gene is amplified and overexpressed in primary medulloblastoma/PNET, and promotes growth of medulloblastoma cell lines. In contrast, NotcM inhibits medulloblastoma cell proliferation. We are the first group to define an in vitro system in which two Notch receptors have opposing effects. We will create chimeras between Notch 1 and Notch2 and exploit this system to determine which domains of Notch differentially regulate proliferation and differentiation (Aim I). This should provide basic insights into the mechanism of Notch signaling, and also help guide our development of new therapies. To effectively treat brain tumors via Notch modulation, it will also be important to understand the mechanisms by which the pathway is turned on, as it must be blocked at or below the level of activation. In Aim II, we will therefore determine the roles of ligand stimulation and receptor mutation in activating the Notch pathway in medulloblastoma. A third aim is to assess the effects of Notch pathway blockade on tumor stems cells and tumor propagation. We have already shown that Notch pathway blockade inhibits proliferation and induces apoptosis of medulloblastoma in vitro. Recently, cells with stem- like properties have been isolated from freshly resected human medulloblastoma/PNET. This subset of neoplastic cells appears to be critical in propagating brain tumors, functioning as "tumor stem cells." Targeted therapies causing tumor stem cells to differentiate or die therefore represent a novel therapeutic avenue with great promise. We hypothesize that the Notch pathway, which controls neural stem cell survival and proliferation during normal fetal development, will also be required for tumor stem cells survival, and will test this in Aim III. Finally, it is not known if persistent activation of Nbtch2 is sufficient to initiate the formation of medulloblastoma. In Aim IV, we will introduce activated Notch2 into cerebellar precursor cells and assess its transforming capacity.